Evaluating the effects of whole genome amplification strategies for amplifying trace DNA using capillary electrophoresis and massive parallel sequencing

Abstract

To draw robust conclusions when trace DNA samples are detected in complex cases, it is essential to successfully recover and genotype short tandem repeats (STRs) from trace DNA. However, obtaining complete STR profiles by the conventional polymerase chain reaction-capillary electrophoresis (PCR-CE) method is generally difficult as trace DNA is often less than 100 pg. Previous studies have proven that through whole-genome amplification (WGA), the yield of DNA from trace DNA samples could be improved. In this study, we used two WGA kits, namely, REPLI-g® Single Cell kit and MALBAC® Single Cell DNA Quick-Amp Kit (hereafter referred to as REPLI and MALBAC), to amplify DNA samples with a series of dilutions (from 5.00 ng/μL to 0.391 pg/μL). Typing of STR markers in samples with and without WGA were then performed on a CE platform by the application of Goldeneye® DNA ID System 20 A kit, as well as directly calling sequences from massive parallel sequencing (MPS) for WGA samples with 1.00 ng, 125 pg and 25.0 pg as DNA inputs. Quantification results demonstrated that the yield of samples with WGA could reach the microgram level. The amplification fold was at least > 2000 and > 200 for REPLI and MALBAC, respectively. CE results showed that the number of correctly called loci was improved for trace DNA after WGA when the DNA inputs were lower than 25.0 pg for REPLI and 6.25 pg for MALBAC, respectively. WGA remarkably improved the percentage of called loci with DNA inputs lower than 50.0 pg, although poor performance in repeatability was observed. MPS results suggested that the correctly called loci calculated by MPS reads were mostly more than those calculated by CE, particularly for those of short length, implying MPS of samples after WGA is worth testing in the future. In conclusion, WGA has the potential usability for forensic trace DNA analysis at the single-cell level with good fidelity, although its repeatability requires further improvement.